Dissecting the roles of TfRs and HSPA5 in Pu accumulation in liver Abstract: Plutonium (Pu) is one of the most toxic of all actinide elements produced in high quantities around the world. Many Pu radioisotopes have very long half-lives and their extended storage leads to risks of eventual contamination that can affect both the clean-up workers and the public. While many studies of interactions between Pu and mammalian cells were done in the past, the cell and molecular biology aspects of these interactions remain unexplored. In addition, technologies for studying such interactions have changed during the past decade. Our team has used unique resources offered by the actinide laboratory and synchrotron at Argonne National Laboratory (ANL) to carry out novel types of experiments using X-ray fluorescence spectroscopy and microscopy that can be done only at third generation synchrotrons such as the Advanced Photon Source (APS) at ANL. We now propose to interrogate Pu interaction with liver cells in 2D and 3D cell cultures, probing several Pu-protein interactions we predict based on our preliminary and published data. For this work we will once again use the facilities at the APS but, in addition to equipment we used in publications that comprise the bulk of the preliminary data in this project, we will also use a newly built X-ray microscopy instrument, the Bionanoprobe also at the APS. This new, custom built instrument can be used to obtain tomographic X-ray fluorescence data on cryogenically prepared Pu-containing specimens in order to validate Pu-protein interactions, quantitate and map Pu in 3D, determine its oxidation state and probe its chemical environment. This work will be complemented by gene expression profiling and protein mass spectrometry of the Pu exposed cells' secretome in the interest of identifying biomarkers of Pu exposure under different physiological conditions.